PROJECT SUMMARY Antibiotic resistance is a serious problem in the United States and around the world, particularly in hospitals. In the U.S. alone, millions of people acquire antibiotic-resistant infections each year, and thousands die from these infections. Although antibiotic resistance can be controlled to some degree by surveillance programs that track resistance and by establishing strict guidelines for appropriate antibiotic use, the natural selection process cannot be stopped and resistance will ultimately emerge. Therefore, it is imperative that we maintain a pipeline of new antibiotics and develop antibiotics that act on novel drug targets. Our project addresses these critical needs by defining the mechanism of a naturally occurring bacteriostatic inhibitor of Escherichia coli. N4gp8, a polypeptide produced by bacteriophage N4, binds the DNA polymerase clamp loader and rapidly shuts down DNA replication by inhibiting clamp loading. Because clamp loaders are absolutely required for DNA replication in all bacteria, and consequently for cell growth and proliferation, the clamp loader represents an attractive, and to date unexploited, drug target. Our overarching hypothesis is that N4gp8 presents a model for the design of a new class of antibiotics that act on a novel target. This project will define the mechanism of action of N4gp8 to inform future efforts to develop clamp loader inhibitors. The clamp loader catalyzes the assembly of ring-shaped sliding clamps on DNA, and in doing so, must bind three ligands, ATP, the clamp, and DNA and undergo ligand-induced conformational changes that drive the mechanical reaction. Therefore, there are multiple mechanisms by which N4gp8 could function. This proposal will define the mechanism of inhibition of clamp loading by N4gp8 by defining which clamp loader activity is inhibited in Aim I and by defining molecular interactions between the N4gp8 and the clamp loader that are required for inhibition activity in Aim II. Assays that we developed to investigate clamp loader mechanism will be used along with standard binding assays to identify the step in the clamp loading reaction that is inhibited by N4gp8 and to measure N4gp8-clamp loader binding constants. A combination of techniques will be used to characterize the solution structure of N4gp8 and its active oligomeric state, as well as to define the binding site for N4gp8 on the clamp loader and key protein-protein interactions required for inhibition. These mechanistic studies will elucidate structure-function relationships that can be used in the rational structure-based design of new clamp loader inhibitors, and in developing screens for inhibitors that function by a N4gp8-type mechanism. The clinical relevance of the E. coli model system used here is high because E. coli is a member of the Enterobacteriaceae family of Gram-negative bacteria, and carbapenem-resistant Enterobacteriaceae are classified by the CDC as an urgent antibiotic resistance threat.